Chimeric nucleic acid molecules with non-aug translation initiation sequences and uses thereof

Abstract

The present disclosure relates to nucleic acid vaccine compositions and methods for preventing or treating pathological conditions, such as cancer or infectious disease. Further, the disclosure provides methods for more efficient production of antigens via mRNA containing one or more non-conventional start codons to promote multiplex initiation of translation in eukaryotic cells.

What is claimed is: 
     
         1 . A chimeric nucleic acid molecule, comprising a multiplex translation initiation (MTI) sequence comprising from two to about five translation initiation sites operatively linked in frame to a nucleic acid molecule encoding a fusion protein comprised of a plurality of polypeptide components, wherein at least one of the MTI translation initiation sites is a non-AUG translation initiation site and the MTI allows the production of more than one mole of fusion protein per mole of mRNA. 
     
     
         2 . The chimeric nucleic acid molecule of  claim 1 , wherein the MTI comprises one, two, three, or four non-AUG translation initiation sites. 
     
     
         3 . The chimeric nucleic acid molecule of  claim 2 , wherein the non-AUG translation initiation sites are CUG translation initiation sites. 
     
     
         4 . The chimeric nucleic acid molecule of  claim 3 , wherein the MTI comprises an AUG translation initiation site downstream of the CUG translation initiation sites. 
     
     
         5 . The chimeric nucleic acid molecule of  claim 1 , wherein the MTI comprises (a) a nucleic acid molecule encoding one or two nuclear localization domains located downstream of two or three CUG translation initiation sites and upstream of an AUG translation initiation site, or (b) two or three CUG translation initiation sites upstream of a nucleic acid molecule encoding one or two nuclear localization domains and no AUG translation initiation site. 
     
     
         6 . The chimeric nucleic acid molecule of  claim 1 , wherein the MTI comprises a 5′-portion of a human FGF2 gene, wherein the 5′-portion of the human FGF2 gene contains an FGF2 AUG translation initiation site and about 123 nucleotides to about 385 nucleotides upstream of the FGF2 AUG translation initiation site that is in frame with the nucleic acid molecule encoding the fusion protein. 
     
     
         7 . The chimeric nucleic acid molecule of  claim 6 , wherein the MTI further comprises from about 15 nucleotides to about 45 nucleotides downstream of the FGF2 AUG translation initiation site. 
     
     
         8 . The chimeric nucleic acid molecule of  claim 6 , wherein the 5′-portion of the human FGF2 gene encodes a polypeptide having at least 90% sequence identity to any one of the amino acid sequences set forth in SEQ ID NOS.:8-12, or encodes a polypeptide as set forth in any one of SEQ ID NOS.:8-12. 
     
     
         9 . The chimeric nucleic acid molecule of  claim 1 , wherein the MTI sequence has at least 90% sequence identity to a nucleotide sequence as set forth in any one of SEQ ID NOS.:1-6, 95, or 96. 
     
     
         10 . The chimeric nucleic acid molecule of  claim 1 , wherein the encoded fusion protein comprises from two to about ten polypeptide components. 
     
     
         11 . The chimeric nucleic acid molecule of  claim 10 , wherein one or more of each encoded polypeptide component of the fusion protein further comprises on the N-terminus and/or C-terminus: (a) from one to about ten junction amino acids; (b) a spacer comprising from two to about 35 amino acids; (c) a spacer comprising a (Gly 4 Ser) n  wherein n is an integer from 1 to 5; (d) a natural cleavage site comprising from one to about ten amino acids; (e) a self-cleaving amino acid sequence; (f) an intracellular trafficking sequence; or (g) any combination thereof. 
     
     
         12 . The chimeric nucleic acid molecule of  claim 10 , wherein the two to about ten polypeptide components each comprise an antigenic peptide. 
     
     
         13 . The chimeric nucleic acid molecule of  claim 12 , wherein two to about ten of the antigenic peptides are each different from each other. 
     
     
         14 . The chimeric nucleic acid molecule of  claim 12 , wherein the antigenic peptide comprises a tumor-associated antigen or a pathogen-related antigen. 
     
     
         15 . The chimeric nucleic acid molecule of  claim 14 , wherein the tumor-associated antigen comprises an antigen associated with breast cancer, triple negative breast cancer, inflammatory breast cancer, ovarian cancer, uterine cancer, colorectal cancer, colon cancer, primary peritoneal cancer, testicular cancer, renal cancer, melanoma, glioblastoma, lung cancer, prostate cancer, or any combination thereof. 
     
     
         16 . The chimeric nucleic acid molecule of  claim 14 , wherein the pathogen-related antigen comprises an antigen from a virus, parasite, or bacteria. 
     
     
         17 . The chimeric nucleic acid molecule of  claim 12 , wherein one or more of the antigenic peptides are an HLA Class I antigenic peptide, an HLA Class II antigenic peptide, an HLA Class II antigenic peptide comprising an embedded HLA Class I antigenic peptide, or any combination thereof. 
     
     
         18 . The chimeric nucleic acid molecule of  claim 1 , wherein the encoded fusion protein comprises an amino acid cleavage sequence amino-terminal to one or more of the encoded polypeptide components of the fusion protein, wherein the amino acid cleavage sequence comprises a 2A peptide from porcine teschovirus-1 (P2A), equine rhinitis A virus (E2A),  Thosea asigna  virus (T2A), or foot-and-mouth disease virus (F2A). 
     
     
         19 . The chimeric nucleic acid molecule of  claim 1 , wherein the encoded fusion protein comprises a secretion signal amino acid sequence, a membrane localization amino acid sequence, an endosome targeting sequence, a dendritic cell targeting amino acid sequence, or any combination thereof. 
     
     
         20 . The chimeric nucleic acid molecule of  claim 1 , wherein the encoded fusion protein comprises:
 (a) a VSVG signal amino acid sequence as encoded by the polynucleotide of SEQ ID NO.:43 that is operably linked in frame to and disposed between the MTI sequence and the nucleic acid molecule encoding the fusion protein, optionally comprising a nucleic acid molecule encoding an amino acid cleavage sequence operably linked in frame to and disposed between the MTI sequence and the nucleic acid molecule encoding the VSVG signal amino acid sequence;   (b) a VSVG trafficking amino acid sequence as encoded by the polynucleotide of SEQ ID NO.:47 or 48 that is operably linked in frame to the MTI sequence;   (c) a VSVG membrane localization amino acid sequence as encoded by nucleotides 457 to 516 of SEQ ID NO.:57 that is operably linked in frame to the MTI sequence;   (d) a VSVG trafficking amino acid sequence and membrane localization amino acid sequence as encoded by the polynucleotide of SEQ ID NO.:57 that is operably linked in frame to the MTI sequence;   (e) any one of (a)-(d) further comprising a dendritic cell targeting amino acid sequence as encoded by the polynucleotide of SEQ ID NO.:45; or any one of (a) or (c)-(e) further comprising an intracellular trafficking sequence.   
     
     
         21 . The chimeric nucleic acid molecule of  claim 1 , wherein the chimeric nucleic acid molecule is:
 (a) an mRNA molecule;   (b) a DNA molecule;   (c) a DNA or an RNA molecule contained in a vector and operably linked to an expression control sequence.   
     
     
         22 . The chimeric nucleic acid molecule of  claim 21 , wherein the vector of subpart (c) comprises a plasmid vector or a viral vector. 
     
     
         23 . The chimeric nucleic acid molecule of  claim 22 , wherein the vector comprises a viral vector selected from a rhabdoviral, adenoviral, herpesviral, poxviral, or retroviral vector. 
     
     
         24 . A composition, comprising a chimeric nucleic acid molecule of  claim 1  and a therapeutically acceptable carrier or excipient. 
     
     
         25 . A method of eliciting an immune response, comprising administering to a mammalian subject a therapeutically effective amount of a chimeric nucleic acid molecule according to  claim 1 , wherein the plurality of polypeptide components comprises from two to about ten antigenic peptides. 
     
     
         26 . The method of  claim 25 , further comprising administering an effective amount of an antigenic peptide immunization composition. 
     
     
         27 . The method of  claim 26 , wherein the chimeric nucleic acid molecule encodes one or more of the same antigenic peptides of the antigenic peptide composition. 
     
     
         28 . The method of  claim 26 , wherein:
 (a) the chimeric nucleic acid molecule and the antigenic peptide immunization compositions are administered simultaneously;   (b) the chimeric nucleic acid molecule and the antigenic peptide immunization compositions are administered sequentially;   (b) the chimeric nucleic acid molecule is administered from 1 hour to 8 weeks after the antigenic peptide immunization composition;   (c) the antigenic peptide immunization composition is administered from 1 hour to 8 weeks after the chimeric nucleic acid molecule.   
     
     
         29 . The method of  claim 28 , further comprising one or more additional administrations of an effective amount of the antigenic peptide immunization composition and/or the chimeric nucleic acid molecule after the first administration of the antigenic peptide immunization composition and/or the chimeric nucleic acid molecule. 
     
     
         30 . The method of  claim 25 , wherein before or after administering the chimeric nucleic acid molecule encoding the fusion protein comprised of a plurality of tumor-associated antigens, the human subject is treated with surgery, chemotherapy, radiation therapy, antibody therapy, immunosuppressive therapy, and any combination thereof 
     
     
         31 . The method of  claim 25 , wherein the first administration is intradermal. 
     
     
         32 . The method of  claim 31 , wherein the one or more additional administrations are intradermal, intravenous, intramuscular, or any combination thereof.